The principal objectives of this research project are six-fold: 1) to determine host and viral factors which influence the genetically controlled resistance of inbred mouse strains to street rabies virus (SRV) 2) to determine if an in vivo laboratory model for persistent rabies virus infection can be established with subsequent goals of determining in which cell(s) the virus (genome) is sequestered and whether the virus can be activated to produce clinical disease 3) to understand the role of macrophages in rabies virus infections 4) to compare the protective capabilities of recombinant vaccines expressing different or multiple structural proteins of the rabies virus against genetic variants of rabies virus that have been isolated worldwide 5) to understand the complex interaction between microglial cells, lymphocytes and cytokines in the pathogenesis of rabies virus infections and 6) to determine the feasibility of using "naked DNA" vaccines to protect mice against a global spectrum of rabies virus variants. We have determined that both tissue culture adapted and non-tissue culture adapted wild strains of rabies viruses replicate in primary murine bone marrow-derived macrophages and murine and human macrophage-like cell lines. Similar viruses also have been shown to replicate in primary murine microglial cells, primary feline and human mixed glial cells, and in primary human astrocytes. Rabies viruses harvested from persistently infected cells were shown to have specifically adapted to each cell type. Furthermore, after extended passage, viruses released from persistently infected macrophages lost virulence as determined by their inability to kill intracranially inoculated mice. Recombinant vaccinia viruses expressing either the glycoprotein (G), or both the G and nucleoprotein (N) (GN) of the challenge virus strain (CVS) of rabies virus protected mice (>95%) against a global spectrum of rabies virus variants. The concurrent expression of G and N in a vaccine was no more protective than the expression of G alone. A recombinant virus expressing only N was less protective. Antibody prepared against the G of the strains used in the vaccines neutralized all variant viruses, and sera from mice infected with any one variant cross-neutralized all of the other viruses. Attempts to isolate infectious virus (genome) from persistently infected mice are ongoing. Preliminary data indicate that RT-PCR is more sensitive than in vitro amplification of infectious virus, particularly in mice which have high titers of serum and brain neutralizing antibody. Furthermore, sets of N and G primers have been prepared which detect all viruses that were used to establish persistent infections. Initial studies have determined that neutralizing antibody is elicited following immunization with "naked DNA" vaccines encoding the CVS G gene. Ongoing studies will establish optimal conditions (route of vaccination, concentration of DNA, eukaryotic expression vector) for protection of mice against a global spectrum of rabies virus variants.